Zhichun Lai

GST FUSIONS FOR ANTIBODY PRODUCTION

pGEX; is a protein expression vector for use in E.coli that carries the GST (glutathione S-transferase, 26 Kd) gene from the parasitic helminth Schistosoma japonicum. A gene encoding a foreign protein is fused at the terminus of the GST gene at a polylinker (BamHI, SmaI, EcoRI). Each of the three vectors, pGEX-1, pGEX-2X and pGEX-3X, generates fusions in a different ORF. The GST gene is under control of the tac promoter and is induced by IPTG. Plasmid derived lacq product is sufficent to inhibt the GST gene expression in the absence of inducers.
Most GST fusion proteins; are soluble, and one may purify these GST fusion proteins by affinity to glutathione;, the substrate of the enzyme, linked to agarose beads. Supernatant containing GST or GST fusion protein is mixed with the beads. After extensive washing, the adsorbed protein is released by adding free glutathione as competitor. This one step purification gives pretty pure material suitable for injection into an animal, or for biochemical analysis. GST fusion proteins are assumed to keep their native comformation, since no denaturation steps are involved.
The yield is about 1.6-15 mg/liter culture.
antibody prduction;
Mini Prep:

1) Screen TG2 transformant.

2) Take 100 µl overnight culture into 900 µl 2xYT with 100 µg/ml ampicillin.

3) Grow at 37°C for 1.5 hours; add IPTG to final concentration of 1.0 mM; grow 1.5 hours at 37°C. Spin down cells, resuspend in 300 µl PBT buffer (PBS + 1% Triton X-100) with 0.2 mg/ml lysozyme; sonicated about 5" (not more than 10"); spin down debris; mix supernatant with 50 µl 50% glutathione-agarose beads; leave at room temperature for a few minutes; wash 3x 1ml PBT; add 30 µl 3x SDS sample buffer and boil for 5 min. before loading to SDS protein gel.

Comments: If you don't see any fusion protein with expected size, you may run total protein on the gel. Use the one without IPTG as a control. It is possible that fusion protein is insoluble. If you still don't see anything, just simply give up, try other constructs, or other expression systems.

Maxi-Prep:

1) Dilute overnight culture 1:10 into LB or 2xYT broth with 100 µg/ml ampicillin. Grow at 37°C until OD600 reaches 0.6-1.0. It takes about 1-3 hours.

2) Add IPTG to final 0.5 mM, continue growth for 2-3 hours.

3) Spin down cells; freeze at -70°C for 5 min.; thaw in 1:100 to 1:50 culture volume of PBS containing 1 mM PMSF, 10 mM -mercaptoethanol, 0.2 mg/ml lysozyme. Leave at 4°C for 20 min..

4) Add Triton X-100 to 1%; sonicate cells for 30-60" (do not over-sonicate; it may cause denaturation. Multiple bands will be seen on the SDS gel because of the association of denatured GST fusion protein with other proteins.)

5) Spin 10,000 x g for 5 min. at 4°C.

6) Mix supernatant with 1 ml 50% glutathione-agarose beads (For 500 ml culture) ; leave at room temperature for 10 min., while slowly shaking; collect beads at 500 x g; wash 3x 50 ml PBS with 1% Triton X-100; elute fusion protein 2x 2 min. with 1 bead volume with freshly made 5 mM reduced glutathione in 50 mM Tris-Cl pH 8.0 buffer.

€50% Glutathione-agarose beads; (Sulphur linkage, Sigma G-4510) is preswollen in PBS + 1% Triton X-100, washed twice and kept at 4°C.

€Beads can be reused after washing in 3 M NaCl.

€Smaller fusion proteins (50kD or less) tend to give better yields; it is also best to avoid regions of th gene that encode hydrophobic peptides.

Reference:

Smith, D. B. and Johnson, K. S. Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene 67:31-40, 1988.